Modulation of mRNA Expression of Monoacylglycerol Lipase, Diacylglycerol Lipase and Cannabinoid Receptor-1 in Mice Experimentally Infected with T. gondii

Toxoplasma gondii, an obligate intracellular parasite, infects more than 30% of world's population. This parasite is considered to be neurotropic, and has high tropism for the central nervous system, and potentially induces cryptogenic epilepsy by no clear mechanism. The current study aimed to investigate the alteration of the main components of the endocannabinoid signaling systems in T. gondii-infected mice. For this purpose, the levels of mRNA expression of monoacylglycerol lipase (MAGL), diacylglycerol lipase (DAGL) and cannabinoid receptor-1 (CB1), were measured by quantitative real time PCR.The mRNA expression level of MAGL was increased by ~ 8-fold in the brains of the Toxoplasma-infected group in comparison with non-infected mice (P<0.0001). The mRNA expression of CB1 gene in the brain of the infected mice was ~ 2 times higher than that measured in control group (P<0.01). The mRNA expression level of DAGL remained unchanged in the infected mice. Overall a substantial increase in MAGL and CB1 expression without any changes in DAGL, in the brain of infected mice suggests that T. gondii disturbs the endocannabinoid signaling pathways, which are known as neurotransmitter modulators involved in epilepsy.

nervous system (CNS) (1). Previous scientific literature has revealed that both acute and chronic infections by T. gondii are associated with neurobehavioral and neurological abnormalities in humans and murine models (1,2). The influence of T. gondii may manifest as different neurological presentations such as epilepsy and seizure (1).
Epilepsy is a common neurological disease that affects over 70 million people worldwide. The main cause of epilepsy is not clear in approximately 60% of patients which is called cryptogenic epilepsy (3,4). One important causative factor for cryptogenic epilepsy is an infectious disease named toxoplasmosis (5). This infection causes convulsions without any other clinical manifestation in chronic phase, while immunosuppressed status converts chronic toxoplasmosis to acute toxoplasmosis and leads to recurrent convulsions (5). Furthermore, systematic review and metaanalysis studies have shown that toxoplasmosis should be considered as a risk factor for epilepsy (6,7). However, the molecular mechanism of the interaction between T. gondii and epilepsy is not clear. It is plausible that the parasite may influence neurological function through alteration in neurotransmitters, receptors, ion channels, and other central components of brain physiology (8).
Modulation in the expression of host genes, and disruption of GABAergic and glutamatergic signaling pathways were observed in T. gondiiinfected mice (9,10). The regulation of these neurotransmitters is affected by other molecules such as cannabinoids which plays an important role in the physiological and pathological processes of epilepsy (11). 2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid that is produced in the CNS. It is synthesized from diacylglycerols (DAG) by diacylglycerol lipase (DAGL). It has been demonstrated that 2-AG acts as a retrograde messenger in CNS by binding to the cannabinoid receptor-1 (CB1) and is finally degraded by monoacylglycerol lipase (MAGL) (12).
Based on the aforementioned evidence regarding the association between T. gondii and epilepsy, and also between epilepsy and endocannabinoids, it was assumed that T. gondii may induce epilepsy through disrupting the cannabinoid pathway. Therefore, the current study was conducted to evaluate the changes in gene expression of MAGL, DAGL and CB1 in mice brains, experimentally infected with T. gondii RH strain.

Toxoplasmosis induction in mice
Tachyzoites of T. gondii RH strain (Babol University Medical Sciences, Babol, Iran) (13) were retrieved from -80°C and injected intraperitoneally to two mice that were then monitored daily to observe abnormal physical signs such as weakness, immobility, and ataxia. The

Statistical analysis
The data was statistically analyzed by

Toxoplasmosis confirmation in infected mice
Clinical symptoms related to toxoplasmosis such as weakness, immobility and ataxia were observed in the inoculated mice two days post infection. No symptoms were seen in control group.
The presence of T. gondii DNA in the brain sample of infected mice was confirmed by PCR analysis of the B1 gene of T. gondii and observation of a 321 bp band on agarose gel (Figure 1).

Toxoplasma-infected mice
As shown in Figure 2, the expression of CB1 gene in the brain of infected mice was~ 2 times higher than that measured in the control group In the present study, we showed that T. gondii disrupts MAGL and CB1 genes expression. It is well known that T. gondii is capable of interfering with the hosts' molecular processes, through either direct interaction or indirect mechanisms (18).
Toxoplasma releases parasite-encoded effector proteins that change the biological system in regions with no tachyzoites or bradyzoites. These changes may occur anywhere in the brain (18 (19).
The amount of MAGL, DAGL and CB1 at protein level, and the expression level of 2-AG were not evaluated in the present study.
In conclusion, this is the first study evaluating

Conflicts of Interest
Authors declare no conflict of interest.